Some protein immobilization methods are conventionally known. In order to industrially utilize enzymes (namely, proteins), some methods for immobilizing protein molecules on various solid supports by forming a bond between a highly reactive side chain of various amino acid residues contained in proteins and a functional group present on a solid support have been developed. Among these methods, widely used methods in which a reaction can be easily caused and a protein molecule can be reacted under gentle conditions without denaturation are an amine coupling method and a surface thiol coupling method.
The amine coupling method is a method in which a carboxyl group precedently immobilized on a solid support is converted into succinimidyl ester by using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide for activation, and a protein is added to be covalently bound thereto via an amino group on the surface of the protein molecule (Non Patent Literature 1).
The surface thiol coupling method is a method in which a thiol group is precedently introduced onto a solid support via a spacer, and an activated protein is added thereto to be covalently bound (Non Patent Literature 2). Specifically, this method is performed as follows: First, a carboxyl group of a protein molecule is allowed to react with 2-(2-pyridinyldithio)ethylamine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, so as to be converted into a derivative having an active disulfide bond. Next, cystamine is added to a solid support having been activated by the same method as in the amine coupling method and subsequently reduced to be derivatized to a thiol group. The precedently derivatized protein is added to this solid support for forming a covalent bond through disulfide exchange, and thus, immobilization is completed.
In both of the amine coupling method and the surface thiol coupling method, a specific chemical group (that is, an amino group (—NH2) in the amine coupling method and a thiol group (—SH) in the surface thiol coupling method) is used for the immobilization, and for immobilizing a protein molecule, the chemical group on an immobilization substrate is reacted with a functional group of a specific amino acid side chain of the protein molecule. Since a typical eucaryote-derived protein molecule has a molecular weight of approximately 50,000, 400 to 500 amino acid residues are contained therein, and accordingly, multiple amino acids of the same type are present in various directions on the surface of the molecule. Therefore, when a protein molecule is immobilized, the molecule orientation is ununiform. Accordingly, a protein may be immobilized in orientation unsuitable to an interaction analysis subsequently performed or an enzyme reaction performed on the solid support in many cases.
In order to avoid such ununiformity in the orientation occurring in the immobilization, an immobilization method utilizing a specific interaction between biopolymers has been developed (Non Patent Literature 3). A system applying an avidin-biotin interaction is put to practical use widely because these molecules are stable and spontaneously bound to each other in the vicinity of a neutral pH. The crystal structure of an avidin-biotin complex has already been elucidated, and the pattern of the interaction at the atomic level has been clarified.
It has been found, based on a three-dimensional structure of a complex in which a biotin molecule is bound to the base protein avidin, that the biotin molecule is bound to a widely exposed region on the surface of the avidin molecule. Therefore, the function of a macromolecule (such as a protein molecule) modified by the biotin molecule is not inhibited by steric exclusion, which is one of reasons why this method has been widely applied. Furthermore, since a biotinylation sequence can be introduced to a specific position in a protein molecule, the molecule orientation can be made uniform in the immobilization of the protein molecule.
In this method, however, it is necessary to modify a target molecule (protein) with biotin prior to the immobilization, and in addition, it is also necessary to precedently immobilize an avidin molecule somehow on a solid support on which the protein is to be immobilized, and thus complicated processes are required. Furthermore, it is necessary to introduce, for biotin modification, an amino acid sequence of more than ten residues into a target protein, which may damage the essential function of the target protein. Moreover, since it is necessary to immobilize a macromolecule like avidin on a solid support, the molecular weight involved in the whole system is increased, and therefore, if it is necessary to measure charge transfer or slight molecular weight change after the immobilization, this method is not suitably employed.
As an immobilization method in which the orientation of a protein molecule on a solid support can be controlled and merely small change in molecular weight is caused by a chemical group introduced into a system, a method utilizing a histidine tag can be employed (Patent Literatures 1 to 3). A histidine tag is a peptide sequence containing 4 to 6 histidine residues, and this site can be used for chelating a metal ion. Since this sequence can be introduced to any position in a protein molecule, the orientation of the protein molecule can be controlled in the immobilization onto a solid support. Furthermore, since the peptide sequence contains 4 to 6 residues and the molecular weight of a cyclic ligand holding a metal ion on the solid support is approximately 200 at most, the increase in molecular weight in the whole system is small.
In the method utilizing a histidine tag, however, since a coordinate bond formed by chelating a metal ion is employed for binding, the bond stability and the bond strength are lower than those of a covalent bond, and hence, the bond is liable to be affected by pH change or ambient salt concentration change and can be easily dissociated. The stability as a solid support for use in protein immobilization is poorer than that attained in the aforementioned three methods.
Besides, a method in which a polypeptide having unnatural amino acid incorporated therein and a molecule having a functional group specifically interacting with a side chain of the unnatural amino acid are used for detecting the polypeptide (Patent Literature 4), and a method in which a polypeptide having unnatural amino acid incorporated therein is attached to a solid support via the unnatural amino acid (Patent Literature 5) to prepare a solid support-binding protein have been reported.
On the other hand, poly((2-methacryloyloxyethyl phosphorylcholine)-co-(n-butyl methacrylate)-co-(p-vinyl phenylboronic acid)) (hereinafter designated as “PMBV”) is a polymer consisting of three units. The respective units are designated as MPC, BMA and VPBA, and play individual roles.
A phosphorykholine group of the MPC unit is known as one of hydrophilic groups present in a lipid bilayer of a cell membrane in vivo. Since a lipid bilayer forms a boundary (cell membrane) of a liquid-liquid interface in vivo, most biopolymers are made not to be adsorbed non-specifically onto this boundary surface. In this manner, the MPC unit prevents a biopolymer such as a protein from being non-specifically adsorbed onto the PMBV polymer, and thus plays a role to improve biocompatibility of a substrate coated with the PMBV polymer (Non Patent Literature 4).
The BMA unit plays a role to suppress too much increase of the polymerization speed in polymer synthesis. Furthermore, since the MPC and the VPBA are extremely hydrophilic (MPC) and hydrophobic (VPBA), if merely the MPC and the VPBA are contained, formation of a granular aggregate having the MPC disposed outside and the VPBA disposed inside is accelerated. Since the BMA has, however, an intermediate hydrophilicity between the MPC and the VPBA, the BMA plays, when added, a role to suppress the formation of the aggregate and to uniformly develop the polymer in an aqueous solution.
The VPBA unit is an active residue having a reaction activity to form a covalent bond with another molecule. The PMBV polymer has a function to increase the biocompatibility of a substrate as a coating agent by itself. When reacted with another polymer, however, it may be processed into a gel or a sheet, so as to be used by itself as a biocompatible substrate. Here, if the PMBV polymer is reacted with a polymer, such as polyvinyl alcohol (PVA), having a flexible structure in which a plurality of —OH groups contained in a molecule can be close to one another within a given distance, a boronic acid group contained in the VPBA is reacted with the plural —OH groups, so that a bivalent covalent bond can be formed. In this manner, the PMBV can be easily processed into the form of a gel or a sheet when reacted with the PVA, and the moisture content and the hardness can be adjusted by changing the composition ratio among the respective units in the PMBV (Non Patent Literature 5).
An MPC polymer containing the MPC as one component is used for coating an artificial vessel or an artificial joint by utilizing its bioinert characteristics owing to a phosphorylcholine group derived from a cell membrane component, and thus, the coated artificial vessel or joint can be used for a long period of time (Non Patent Literatures 6 and 7). By using its property of being easy to form a gel through a reaction with the PVA or the like, application to a technique to independently culture individual cells within a gel has started (Non Patent Literature 5). This technique is expected as indispensable elemental technology for single cell analysis presumed to be widely spread in the future.